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As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)–encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8 + T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

Pediococcus pentosaceus LBM 18 has shown potential as producer of an antibacterial and antifungal bacteriocin-like inhibitory substance (BLIS). BLIS inhibited the growth of spoilage bacteria belonging to Lactobacillus, Enterococcus and Listeria genera with higher activity than Nisaplin used as control. It gave rise to inhibition halos with diameters from 9.70 to 20.00 mm, with Lactobacillus sakei being the most sensitive strain (13.50–20.00 mm). It also effectively suppressed the growth of fungi isolated from corn grain silage for up to 25 days and impaired morphology of colonies by likely affecting fungal membranes. These results point out that P. pentosaceus BLIS may be used as a new promising alternative to conventional antibacterial and antifungal substances, with potential applications in agriculture and food industry as a natural bio-controlling agent. Moreover, cytotoxicity and cell death induction tests demonstrated cytotoxicity and toxicity of BLIS to human colon adenocarcinoma Caco-2cells but not to peripheral blood mononuclear cells, with suggests possible applications of BLIS also in medical-pharmaceutical applications.

Among skin cancers, melanoma has the highest mortality rate. The heterogeneous genetic melanoma background leads to a tumor-propagating capacity particularly important in maintaining therapeutic resistance, and tumor recurrence. The identification of efficient molecules able to control melanoma progress represents an important opportunity for new therapeutic strategies, particularly in combination with the current standard-of-care treatments. In this context, several studies have reported the antitumor effects of melatonin against different types of cancer, including melanoma. Here, we describe the underlying mechanisms associated with melatonin’s activity in human melanoma cell lines, focusing on cell cycle and cytoskeleton remodeling. Interestingly, while melatonin induced melanocyte DNA replication, melanoma cells exhibited cell cycle arrest in the G1-phase. This phenomenon was associated with cyclin-D1 downregulation or p21 overexpression. The efficacy of melatonin on melanoma cells survival and proliferation was detected using the clonogenic assay, with a decrease in both the number and size of colonies. Additionally, melatonin induced a dramatic cytoskeleton remodeling in all melanoma cell lines, leading to a star-like morphology or cell swelling. The role of melatonin on melanoma cytoskeleton was associated with the actin disruption, with thinning and/or broken actin fibers, and weak and/or loss of paxillin along stress fibers. These data support the observed findings that melatonin impairs melanoma invasion in skin reconstructed models. Together, our results suggest that melatonin could be used to control melanoma growth and support basic and clinical studies on melatonin as a promising immunometabolic adjuvant for melanoma therapy.

Escherichia coli and Shigella are common diarrhea-causing pathogens in children and adults. Enteroinvasive Escherichia coli (EIEC) shares a similar pathogenic mechanism with Shigella. However, EIEC are less virulent than Shigella. The aim of this work was to get a better understanding of the virulence differences between EIEC and S. flexneri. We investigated i) the bacterial gene co-expression networks (GCNs) and ii) the the transcriptional modules (WGCNA) of Caco-2 cells infected with EIEC or with S. flexneri during a three-hour period of bacterial infection. The GCN analysis showed that EIEC and S. flexneri networks presented different topologies. Additionally, the EIEC network revealed that pINV genes are not connected with chromosomal genes. WGCNA and eigengene analysis showed enterocyte gene expression variation along the three-hour bacterial post-infection period. Additionally, at one-hour post-infection EIEC induced a higher number of gene expression changes in Caco-2 cells than S. flexneri. Several of these genes are involved in autophagy. This study showed that the lower virulence of EIEC is associated with a lack of functional cooperation between pINV and chromosomal genes, differently from what was observed in S. flexneri. Consequently, EIEC becomes less efficient in subverting host-cell bacterial recognition as well as defense mechanisms such as autophagy.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation., GRAPHICAL ABSTRACT

The “Adopt a Bacterium” project is based on the use of social network as a tool in Microbiology undergraduate education, improving student learning and encouraging students to participate in collaborative learning. The approach involves active participation of both students and teachers, emphasizing knowledge exchange, based on widely used social media. Students were organized in groups and asked to adopt a specific bacterial genus and, subsequently, submit posts about “adopted genus”. The formative assessment is based on posting information on Facebook®, and the summative assessment involves presentation of seminars about the adopted theme. To evaluate the project, students filled out three anonymous and voluntary surveys. Most of the students enjoyed the activities and positively evaluated the experience. A large amount of students declared a change in their attitude towards the way they processed information, especially regarding the use of scientific sources. Finally, we evaluated knowledge retention six months after the end of the course and students were able to recall relevant Microbiology concepts. Our results suggest that the “Adopt a Bacterium” project represents a useful strategy in Microbiology learning and may be applied to other academic fields.

Considering the great lethality and sequels caused by meningitis, rapid diagnosis and prompt treatment initiation have a great impact on patient outcome. Here, we developed a multiplex-PCR for simultaneous detection of the four most prevalent bacterial pathogens directly in CSF samples. The multiplex-PCR was designed to detect the following genes: fbsA (Streptococcus agalactiae), lytA (Streptococcus pneumoniae), crtA (Neisseria meningitidis), p6 (Haemophilus influenzae), and 16S rRNA (any bacterial agent). The multiplex-PCR showed a DNA detection limit of 1 pg/μL. Among 447 CSF samples tested, 40 were multiplex-PCR positive, in which 27 and 13 had positive and negative bacterial culture, respectively. Our multiplex-PCR is fast, reliable, and easily implementable into a laboratory routine for bacterial meningitis confirmation, especially for patients who previously started antimicrobial therapy. Our molecular approach can substantially improve clinical diagnosis and epidemiological measures of meningitis disease burden.

Cyanide is a toxic compound that is converted to the non-toxic thiocyanate by a rhodanese enzyme. Rhodaneses belong to the family of transferases (sulfurtransferases), which are largely studied. The sulfur donor defines the subfamily of these enzymes as thiosulfate:cyanide sulfurtransferases or rhodaneses (TSTs) or 3-mercaptopyruvate sulfurtransfeases (MSTs). In Mycobacterium tuberculosis, the causative agent of tuberculosis, the gene Rv0815c encodes the protein CysA2, a putative uncharacterized thiosulfate:cyanide sulfurtransferase that belongs to the essential sulfur assimilation pathway in the bacillus and is secreted during infection. In this work, we characterized the functional and structural properties of CysA2 and its kinetic parameters. The recombinant CysA2 is a α/β protein with two rhodanese-like domains that maintains the functional motifs and a catalytic cysteine. Sulfurtransferase activity was determined using thiosulfate and 3-mercaptopyruvate as sulfur donors. The assays showed Km values of 2.89 mM and 7.02 mM for thiosulfate and 3-mercaptopyruvate, respectively, indicating the protein has dual activity as TST and MST. Immunological assays revealed that CysA2 interacted with pulmonary cells, and it was capable to activate macrophages and dendritic cells, indicating the stimulation of the immune response, which is important for its use as an antigen for vaccine development and immunodiagnostic.

The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.

Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8+ T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.

Cervical cancer is a major public health problem and one of the leading causes of cancer deaths in women. Virtually all cases of cervical cancer, as well as a growing share of anal and head/neck tumors, are associated with human papillomavirus (HPV) infection. Despite the effectiveness, the available prophylactic vaccines do not benefit women with cervical lesions or cancer. Therefore, the search of new immunotherapeutic approaches to treat HPV-induced tumors is still a priority. The present study characterizes a therapeutic antitumor vaccine based on the genetic fusion of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) with the E7 oncoprotein from HPV-16 (gDE7). Two subcutaneous doses of gDE7, admixed with poly (I:C), conferred complete and long-lasting therapeutic antitumor protection on mice previously challenged with tumor cells expressing the HPV-16 oncoproteins. The vaccine induced multifunctional E7-specific CD8+ T cells with cytotoxic activity and effector memory phenotype (CD44+ CD62Llow). In addition, gDE7 admixed with poly (I:C) vaccination controlled the expansion of tumor-induced regulatory T cells and myeloid-derived suppressor cells. More importantly, gDE7 activated mouse CD11c+ CD8α+ and human BDCA3+ dendritic cells (DC), specialized in antigen cross-presentation to CD8+ T cells, under in vitro conditions. These results indicated that the activation of a specific DC population, mediated by gD, improved the antigen-specific immune responses and the therapeutic performance induced by antitumor vaccines. These results open perspectives for the clinical testing of gDE7-based vaccines under the concept of active immunization as a tool for the therapeutic control of cancer. Mol Cancer Ther; 16(9); 1922–33. ©2017 AACR.

Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies.

Neste estudo, mostramos a possibilidade de identificação dos AgH de ETEC pela caracterização molecular do gene fliC pela análise de seu polimorfismo de restrição. Um único alelo de fliC de ETEC foi encontrado para cada antígeno flagelar, utilizando-se a endonuclease RsaI, com exceção do H21. Além de cepas móveis, isolados imóveis também puderam ser caracterizados por essa técnica molecular. A alta tipabilidade da PCR-RFLP foi comprovada por meio de sua aplicação não só a amostras de ETEC com AgH previamente desconhecidos, mas também a outras linhagens de E. coli. Observamos que após identificação do antígeno flagelar das amostras de ETEC pela PCR-RFLP, a determinação do antígeno somático pôde ser direcionada, diminuindo assim, o número de anti-soros utilizados para a pesquisa do AgO. A técnica de PCR-RFLP, em nosso estudo, mostrou uma sensibilidade de 83% e 100% de especificidade. Esta técnica foi mais rápida na identificação do AgH de E. coli (2 dias) em comparação à sorologia clássica (7 ou mais dias, dependendo da motilidade da cepa). Acreditamos que o método de soroaglutinação para determinação do AgH será substituído rapidamente pela PCR-RFLP. Contudo, a soro aglutinação não poderá ser totalmente dispensada em curto prazo. No futuro, com o perfil molecular obtido dos alelos de cepas procedentes de estudos epidemiológicos, novos padrões serão definidos para as cepas diarreiogênicas de E. coli, permitindo o abandono da sorologia para AgH. In this study, we showed that the H antigens of ETEC can be characterised by restriction analysis of the polymorphism of the fliC gene. Only one allele of the fliC gene of ETEC was found for each flagellar antigen when restriction endonuclease RsaI was used, with the exception of H21 . Additionally, non-motile strains could also be characterised using this molecular technique. The high typeability of this technique was demonstrated by the fact that it can not only be applied to ETEC samples with previously unknown H antigens but also to all other lineages belonging to the E. coli species. Moreover, the determination of the somatic antigen was guided by the identification of the flagellar antigen of ETEC strains by PCR-RFLP, thus reducing the number of anti-AgO sera used. In this study, the PCR-RFLP technique showed a sensitivity of 83% and a specificity of 100%. This technique proved to be quicker for the identification of the E. coli AgH, taking 2 days to complete, in comparison to the 7 or more days necessary when using classic serotyping. We believe that the determination of the AgH by seroagglutination will soon be substituted by the PCR-RFLP technique. However, serotyping will still have to be used in the short run, for further studies involving PCR-RFLP must be carried out. In the future, with the determination of the molecular profiles of alleles of strains obtained in epidemiological studies, new patterns will have been described for the diarrhoeagenic strains of E. coli, thus permitting the abandonment of AgH serotyping for good.

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coli strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type.

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and biochemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

The activity of the enzyme indoleamine 2,3 dioxygenase-1 (IDO) has been associated with the maintenance of the immunosuppressive microenvironment in many types of cancers. IDO catalyzes the degradation of tryptophan via the kynurenine pathway, which impacts on the effector activity of immune cells. The role of IDO in immune tolerance observed in human papillomaviruses-associated tumors is poor understood. Here, we targeted IDO to improve the efficacy of a therapeutic protein vaccine against HPV-16-associated tumors. The combination of IDO inhibitors with immunotherapy showed evidence that can support a new antitumor therapeutic approach. In the murine model C57BL/6, different biologic parameters were evaluated in order to determine the activation of the protective immunity obtained by the combined immunotherapy. We observed that isoforms of 1-methyl tryptophan, combined with our therapeutic vaccine, led to an activation of cytotoxic CD8 (+) T cells in mice grafted with tumor cells expressing the HPV-16 oncoproteins. In addition, the combination of melatonin enhanced the efficacy of the immunotherapeutic approach, resulting in the complete eradication of tumor masses. Altogether, our results indicate that blocking the immunosuppressive mechanisms mediated by IDO may improve the antitumor response induced by immunotherapies. Citation Format: Ana Carolina Ramos Moreno, Bruna F. M. M. Porchia, Patrícia da Cruz Souza, Roberta Liberato Pagni, Rafael Pegoraro, Luís Carlos de Souza Ferreira. Targeting indoleamine 2,3 dioxygenase to improve the efficacy of a therapeutic vaccine against human papillomaviruses-associated tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B55.

OBJECTIVES: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. This study aimed to investigate the effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation in a live Escherichia coli-induced sepsis model in rats. METHODS: Male Wistar rats were administered an intravenous suspension of E. coli bacteria or were subjected to a sham procedure. Three hours after bacterial infusion, the rats were randomized into the following groups: a control group without treatment, a group treated with lactated Ringer’s solution (4 mL/kg, i.v.), and a group treated with lactated Ringer’s solution (4 mL/kg, i.v.) plus ethyl pyruvate (50 mg/kg). At 24 h after bacterial infusion, leukocyte-endothelial interactions were investigated using intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule-1 was evaluated via immunohistochemistry. White blood cell and platelet counts were also determined at baseline and 3 h and 24 h after E. coli inoculation. RESULTS: The non-treated and lactated Ringer’s solution-treated groups exhibited increases in the numbers of rolling leukocytes (∼2.5-fold increase), adherent cells (∼3.0-fold), and migrated cells (∼3.5-fold) compared with the sham group. In contrast, treatment with Ringer’s ethyl pyruvate solution reduced the numbers of rolling, adherent and migrated leukocytes to the levels observed in the sham group. Additionally, the expression of P-selectin and intercellular adhesion molecule-1 was significantly increased on mesenteric microvessels in the non-treated group compared with the sham group (p

Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4+ and CD8+ T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4+ T lymphocytes from HIV chronic infection and in CD8+ T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.

In this study, we performed fliC PCR-restriction fragment length polymorphism (RFLP) to investigate whether this technique would be better than classic serotyping for the characterization of the H antigen in enterotoxigenic Escherichia coli (ETEC) strains. We showed that the fliC genes from ETEC strains can be characterized by restriction analysis of their polymorphism. Only one allele of the fliC gene from ETEC strains was found for each flagellar antigen, with the exception of H21. Nonmotile strains could also be characterized using this molecular technique. Moreover, determination of the somatic antigen was guided by the identification of the flagellar antigen from previously unknown serotypes of ETEC strains by PCR-RFLP, thus reducing the number of anti-antigen O sera used. The PCR-RFLP technique proved to be faster than classic serotyping for the characterization of the E. coli H antigen, taking 2 days to complete instead of the 7 or more days using classic serotyping. In conclusion, the H molecular typing for Enterobacteriaceae members may become an important epidemiological tool for the characterization of the H antigen of E. coli pathotypes. The PCR-RFLP technique is capable of guiding the determination of the H antigen and could partially replace seroagglutination. With the determination of the molecular profiles of alleles from strains obtained in epidemiological studies, new patterns will be described for ETEC strains or other E. coli pathotypes, thus permitting widespread use of this technique to characterize fliC genes and determine the H antigen of E. coli strains.

Indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO1 is a leading target for interventions aimed at restoring melanoma immune surveillance. Here, in a scenario involving the tryptophan catabolism, we report that melatonin biosynthesis is driven by 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO1, in human fibroblasts, melanocytes and melanoma cells. In addition to melatonin biosynthesis, 1-MT induced the expression of tryptophan hydroxylase, arylalkylamine-N-acetyltransferase and hydroxyindole O-methyltransferase mRNA in fibroblasts and melanocytes. We observed a great variability in the levels of IDO1 mRNA expression and kynurenine release between skin cells and melanoma cell lines in response to interferon-γ, a classical IDO1 inducer. In this setting, melatonin was shown to downregulate kynurenine production. Furthermore, in a condition of low basal activity of IDO1, it was observed that 1-MT, as well melatonin, inhibited the proliferation of human melanoma cells. Taken together, our results suggest that 1-MT may serve as more than just a tool to disrupt tumor immune escape (via the inhibition of IDO1) because it was shown to act directly on the proliferation of human melanoma cells and induce melatonin biosynthesis in the tumor milieu. Moreover, 1-MT-mediated inhibition of IDO occurs in normal skin and melanoma cells, which addresses the possibility that all cells in the skin microenvironment can be targeted by 1-MT. Our findings provide innovative approaches into understanding tumor therapy related to the control of tryptophan metabolism by 1-MT.

We examined the effects of hypertonic saline (HS) on inflammatory, metabolic variables, and bacterial translocation (BT) in rats submitted to intestinal obstruction and ischemia (IO). Male Wistar rats were submitted to IO and treated, 2 h thereafter, with lactated Ringer's (LR) (4 mL/kg per 5 min, i.v.) or HS (7.5% NaCl, 4 mL/kg per 5 min, i.v.). Twenty-four hours after IO, rats were also submitted to enterectomy/enteroanastomosis to resection of necrotized small bowel. Leukocyte-endothelial interactions were investigated by intravital microscopy and the expression of P-selectin and intercellular adhesion molecule 1 by immunohistochemistry. Bacterial cultures of mesenteric lymph nodes, liver, spleen, and blood were used to evaluate BT. Levels of chemokines (cytokine-induced neutrophil chemoattractants 1 and 2), insulin, and corticosterone were determined by enzyme-linked immunosorbent assay. Intestinal histology, serum urea and creatinine levels, and hepatic enzymes activities were performed to evaluate local and remote damage. Relative to IO and LR-treated rats, which exhibited increases in the number of rolling (1.5-fold), adhered (3.5-fold) and migrated (9.0-fold) leukocytes, and increased expression of P-selectin (3-fold) and intercellular adhesion molecule 1 (3-fold) on mesenteric microcirculation, treatment with HS followed by enterectomy reduced leukocyte-endothelial interactions and expression of both adhesion molecules to values attained in sham rats. Serum chemokines were normalized after treatment with both solutions followed by enterectomy. Hypertonic saline-treated rats demonstrated a significant reduction in BT to 50% in liver and spleen samples and bacteremia (14%), compared with 82% of BT in liver and spleen samples of IO and LR-treated rats and bacteremia (57%). Local intestinal damage was attenuated, and renal and hepatic function preserved by treatment with HS followed by enterectomy. Survival rate increased to 86% up to 15 days. Data presented suggest that HS solution followed by enterectomy reduces mesenteric microcirculatory dysfunctions and BT, attenuating local and remote damage in a model of strangulated small bowel obstruction.

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia.Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250-350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured.About 86% of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57% had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (approximately 2-fold), adherent (approximately 5-fold), and migrated leukocytes (approximately 11-fold); this increase was accompanied by an increased expression of P-selectin (approximately 2-fold) and intercellular adhesion molecule-1 (approximately 2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83% at 72 h vs. 0% in Sham rats).Intestinal obstruction and ischemia in rats is a relevant model for the in vivo study of mesenteric microcirculatory dysfunction and the occurrence of bacterial translocation. This model parallels the events implicated in multiple organ dysfunction (MOD) and death.

Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-α, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença. In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-α, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them.

Xanthomonas citri (X. citri), causador do cancro cítrico, afeta muitas áreas de cultivo de citros com impacto comercial. Transportadores ABC foram relatados como essenciais para a patogênese de X. citri. O gene nrtT codifica a proteína periplasmática responsável pelo suposto transporte de nitrato/nitrito/taurina. Neste trabalho, mostramos que a deleção de nrtT não afeta o crescimento de X. citri, porém reduz e atrasa os sintomas do cancro durante a infecção em folhas de citros. Observamos redução na produção de goma xantana e capacidade de aderência na linhagem mutante. A proteína NrtT foi expressa monomérica e monodispersa. Foram observadas alterações na estrutura secundária e aumento da estabilidade térmica de NrtT na presença de MOPS, indicando ser este um possível ligante de NrtT. A proteína PotF descrita como ligadora de putrescina não sofre alteração significativa na estabilidade térmica na presença de putrescina, entretanto, dados de SAXS mostram alterações na estrutura possivelmente decorrentes da ligação com putrescina. Xanthomonas citri (X. citri), which causes citrus canker, affects many citrus growing areas with commercial impact. ABC transporters have been reported as essential for the pathogenesis of X. citri. The nrtT gene encodes the periplasmic protein responsible for the alleged transport of nitrate/nitrite/taurine. In this work, we show that nrtT deletion does not affect the growth of X. citri, but delays and reduces the symptoms of cancer during infection of citrus leaves. We observed a reduction in the production of xanthan gum and adhesion capacity in the mutant strain. The NrtT protein was expressed monomeric and monodisperse. There were changes in the secondary structure and increased thermal stability NrtT in the presence of MOPS, indicating that this was a possible binder NrtT. The PotF protein described as putrescine-binding is not significantly altered thermal stability in the presence of putrescine, however, SAXS data shows changes in the structure possibly resulting from connection with putrescine.